DE3330003A1 - DNA SEQUENCE AND STRUCTURES AND METHOD FOR THE PRODUCTION OF PENICILLIN ACYLASE THEREOF - Google Patents

Description

THOMAS-WIMMER-RINQ 14 D-8000 MUNICH 22

7-

DlPU-CHEM. DR HANS D. BOETERS DJPL-INQ. ROBERT BAUER

PAe BOETERS. BAUER & PARTNER THOMAS-WIMMER-RINQ 14. D-6000 MUNICH 22 MUNICH

DIPHNG. VINCENCE V. RAPFAY

German Patent Office cxpl-chem. dr. thomas fleck

HAMBURG

TELEPHONE: (066) 22 OO 92

8000 Munich 2

TELEGRAMS: PROVENTION. MUNICH

August 19, 1983

Applicant: Society for Biotechnological

Research mbH (GBF), 3300 Braunschweig-Stöckheim

DNA sequence and DNA structures and a method for producing penicillin acylase using them.

It is known that various microorganisms produce penicillin amidase EC 3.5.1.11 or penicillin acylase and that the penicillin acylase gene cloned from E. coli ATCC 11105, for example and is available as 5K (pHM12) = DSM 1610 = NCIB 11670 (DE-OS 29 30 794.6). According to Rothstein et al, Cold Spring Harbor Symposia on quantitative Biology, Vol. XLV (1981) 99-105, is the localizability of a gene by insertion inactivation of a transposon known. DNA sequencing is according to Maxam & Gilbert, Methods Enzymology 65 (1980) 499-560, known. Protein sequencing is known from Laursen, Methods Enzymology 25 (1972) 344-359. The targeted coupling of a gene to a strong possibly inducible promoter is according to Pribnow, Biological Regulation and Development, Vol. 1, 231-277, Plenum Press No. 4 (Goldberger et al. 1979).

It is now an object of the invention to find the gene for penicillin acylase to localize on one of the clones and thus to create the conditions for targeted strain improvement.

The subject of the invention is on the one hand a DNA sequence which is characterized by the following features:

(a) the DNA sequence encodes one of the components of penicillin acylase, which - from an approx. 90k preform - in the ripe state, especially in E.coli, a 2-component aggregate represents, in the form of an approx. 20k subunit (approx. 20 kD) of the following amino-terminal protein sequence:

H-N-GIu-GIn-Ser-Ser-Ser-GIu-Ile-Lys-Ile-Val-Arg-Asp- etc. GAG CAG TCG TCA AGT GAG ATA AAG ATT GTT CGC GAT etc.

or the DNA sequence encodes only part of this component of penicillin acylase,

(b) where the DNA sequence according to (a) may optionally be connected after a promoter or a promoter / leader can,

(c) where the DNA sequence can be obtained from ATCC 11105,

and DNA sequences analogous to (a) which can be obtained from microorganisms, in particular E. coli strains, penicillin acylase produce, these analogous DNA sequences optionally having the feature (b).

The invention also relates to a DNA sequence which is characterized by the following features:

(d) the DNA sequence encodes the other component of penicillin acylase in the form of an approx. 66k subunit of the following amino terminal protein sequence:

7 ^Ί '

H ₂ N-SeΓ-Asn-Met-Trp-Val-IIe-GIy-Lys-SeΓ-Lys-Ala-Gln-Asp- etc, AGC AAT ATG TGG GTG ATC GGC AAA AGC AAA GCC CAG CAT etc.

or the DNA sequence encodes only part of these others Component of penicillin acylase,

(e) where the DNA sequence according to (d) can optionally be preceded by a promoter or a promoter / leader,

(f) where the DNA sequence can be obtained from ATCC 11105,

and DNA sequences analogous to (d) which can be obtained from microorganisms, in particular E. coli strains, which produce penicil- ^s lin acylase, these analogous DNA sequences optionally having feature (e).

The invention also relates to a DNA sequence (total DNA sequence) , which is characterized by the following features:

(g) a DNA sequence (DNA sub-sequence) according to (a),

(h) a DNA sequence (DNA sub-sequence) according to (d), which (read from AGC) follows the DNA sequence according to (a); (i) that the DNA sequence (DNA sub-sequence) according to (a), if appropriate can be connected behind a promoter or promoter / leader according to (b), which for both DNA subsequences responsible for,

as well as DNA sequences (complete DNA sequences) which are analogous to and from the DNA sequence with the features (g), (h) and optionally (i) Microorganisms can be obtained (e.g. Bacillus) that produce penicillin acylase,

as well as DNA sequences (total DNA sequences), which comprise DNA sub-sequences analogous to (g) and (h) and from microorganisms, E. coli strains in particular can be obtained, the penicillin acylase produce, these analogous DNA subsequences

_ A / -. AO .

comprehensive DNA sequences, if applicable, the feature (i) exhibit.

The DNA sequence (total DNA sequence) with the features (g), (h) and (i) can
E. coli include.

3
(h) and (i) can be approx. 2.6 χ 10 base pairs (2.6 kb) for example

For promoters see, for example, Pribnow, loc. cit. For For example, see leader and promoter / leader combinations Inouye & Halegoua, Crit. Rev. Biochem., (1980) 339-371. Leaders ensure that expressed protein from the cytoplasmic area migrate into the periplasm or out of the cell can.

For example, a promoter and a promoter / leader combination according to scheme 1 can be isolated from ATCC 11105:

- the promoter ends with ACA (Thr) and extends about 300 base pairs to the left;

- the leader starts with ATG (Met) and ends with GCT (AIa).

The invention also relates to clonable DNA structures (vectors) in which the said inventive DNA sequences are included.

The invention further provides a process for the production of penicillin acylase, which is characterized in that man

- From a DNA sequence with the features (g) and (h) and obtainable from ATCC 11 105 the promoter or promoter / leader exchanged according to scheme 1 for another promoter or promoter / leader and

- with the help of the exchange product a prokaryotic Allows microorganism to form penicillin acylase.

Instead of the DNA sequence that can be obtained from ATCC 11 105 with the Features (g), (h) and (i) can be incorporated into the inventive Method for this use analogous DNA sequences which have been obtained from microorganisms, in particular E. coli strains that produce penicillin acylase, being the promoters or promoters / leaders of the DNA sequences obtained exchanged for other promoters or promoters / leaders.

The invention further provides a process for the production of penicillin acylase, which is characterized in that man

- The DNA sequence according to (a), which codes only the approx. 20k subunit, behind a possibly inducible promoter or promoter / leader switches,

- The DNA sequence according to (d), which codes only the approx. 66k subunit, behind a possibly inducible promoter or promoter / leader switches and

- the two subunits of a prokaryotic microorganism can form (each individual of the microorganism forming both subunits) and optionally extracted or

- form the two subunits separately in different individuals of a prokaryotic microorganism leaves, optionally extracted and, if necessary, then combines and

- If necessary, the penicillin acylase formed is then isolated.

By switching the DNA sequence according to (a) and according to (d) behind a promoter, increased yields can be achieved to reach penicillin acylase.

Instead of the DNA sequences according to (a) and (d), DNA sequences analogous to this can be included in the method according to the invention.

put that from microorganisms, especially E. coli strains that produce penicillin acylase.

A promoter or promoter / leader can be used in the Method use the DNA sequences obtained from ATCC 11105 according to scheme 1.

The person skilled in the art is familiar with prokaryotic microorganisms (e.g. Bacillus and E. coli), which can be used for the method according to the invention.

It is advantageous in the method according to the invention as prokaryotic microorganism (host strain) to use a strain exhibiting protease activity similar to that of E. coli equals 5K or greater. An example of an E. coli 5K strain is DSM 1454. For protease-active E. coli strains see also Date & Wickner, Proc. Natl. Acad. May be. United States 78 (1981) 6106-6110. By such strains the leader is better split off and possibly that of two components existing protein is broken down better.

The invention is further illustrated by the following examples explained.

example 1

The penicillin acylase was isolated as an aggregate using 5K (pHM12). The subunits 20k and 66k were separated by HPLC chromatography and amino acids from the NHp terminus of both subunits were determined. 20k has the following amino acids:! -! "N-Glu-Gln-Ser-Ser-Ser-Glu-Ile- -Lys-Ile-Val-Arq-Asp-. 66k has the following amino acids: H N-Ser- -Asn-Met-Trp-Val-Ile-Gly-Lys-Ser-Lys-Ala-Gln-Asp-.

Example 2

The EcoRI-HindIII fragment was isolated from pHM12 and the DNA sequence determined. Read from the HindIII interface in the direction of the EcoRI interface it reads as follows:

A AGC TTC GTT GCT AGT ATC AAT TCG CTA ATT ATA CAC CTG CCA GAG GAT ACA * ATG AAA AAT AGA AAT CGT ATG ATC GTG AAC TGT GTT ACT

-Met-Lys-Asn-Arg-Asn-Arg-Met-Ile-Val-Asn-Cys-Val-Thr-GCT TCC CTG ATG TAT TAT TGG AGC TTA CCT GCA CTG GCT * GAG CAG AIa-Se r-Leu-Met-Tyr-Tyr-Trp-Ser-Leu-Pro-Ala-Leu-Ala-Glu-Gln

* ATG: end of promoter and beginning of leader * GAG: end of leader and beginning of the approx. 20k subunit

It codes for the beginning of the 20k subunit. Previous Triplets code for a precursor sequence that does not appear in the mature protein. This is the beginning of the The gene for penicillin acylase is exactly determined so that the prerequisite has been created to target the gene behind to switch a strong and possibly inducible promoter to microorganisms with increased acylase productivity to construct.

Example 3

The complete penicillin acylase gene is isolated from pHM12. The gene is according to Example 1 of DE-PS 28 14 039.8 transferred into E. coli and cloned. The DNA is with Hindlll cut. Thereafter, the DNA sequence encoding the 66 kD subunit is placed behind the promoter / leader sequence according to Scheme 1 switched. The two DNA sequences which encode the two enzyme subunits are separated according to the example 1 of DE-PS 28 14 039.8 transferred to E. coli and

- 8th -

cloned. The separately formed enzyme subunits are extracted and then put together. The yield of penicillin acylase in a 1 liter reactor is 0.1 g.

Example 4

DNA is obtained from Bacillus megaterium ATCC 14 945 and cut with Hpal, EcoRI + Pstl, Ncol on an agarose gel separated, transferred to nitrocellulose paper and hybridized with radioactively labeled penicillin acylase gene from pHM12. DNA regions are homologous to the penicillin acylase gene visible as defined bands on the X-ray film. So the penicillin acylase gene from Bacillus megaterium is that Penicillin acylase gene from pHM12 analogous.

Scheme 1

TCTTT T "AGATICACATTAATG! IAAATTTTTGTIATCAAAAATTIAGTTATCGCG!

! CTCACAGTT CJA TAATGAAAC

! AATTCTCTG CIA AATAGATA ΑΙΑ CC C ₁ AAGCT TIC

! GTTGCTAGTAiTCAATTCG CTiAATTATACAC!

-Leu- -Pro- -GIu- -Asp- -Thr- -Met- -Las- -Asn- -Arä- -Asn-CTGCCAGAG Gi ATACA HTGAAiAAATAGAAAT!

End of promoter and start of leader

-ARä MEt- -ILe VAl ASn CYs- -VAL THR ALa SEr-

CGTATGATC GIT GAACTGTG TIT ACTGCTTCC!

-LEu- -MEt- -TYr- -Tar- -TR »= · - -Ser- -Leu- -Pro- -als- -leu- -ala- ! C TGATGTAT TIA TTGGAGCT TiA CCTGCACT GIG C T

Claims (11)

Claims characterized by the following (a) the DNA sequence encodes one of the components of penicillin acylase in the form of an approx. 20 kD subunit of the following amino terminal protein sequence: HpN-Glu-GIn-Ser-Ser-Ser-GIu-Ile-Lys-Ile-Val-Arg-Asp- etc. GAG CAG TCG TCA AGT GAG ATA AAG ATT GTT CGC GAT etc., or the DNA sequence encodes only part of this component of penicillin acylase, (b) where the DNA sequence according to (a) may optionally be connected after a promoter or a promoter / leader can, Cc) where the DNA sequence can be obtained from ATCC 11105, as well as DNA sequences analogous to (a), which are derived from microorganisms, E. coli strains in particular can be obtained, the penicillin acylase produce, these analogous DNA sequences optionally having the feature (b). -2- 2. DNA sequence, characterized by the following features: (d) the DNA sequence encodes the other component of penicillin acylase in the form of an approx. 66 kD subunit of the following amino terminal protein sequence: '-Asn-Met-Trp-Val-Ile-GIy-Lys-Ser-Lys-Ala-Gln-Asp- etc, AGC AAT ATG TGG GTG ATC GGC AAA AGC AAA GCC CAG CAT etc. or the DNA sequence encodes only part of this other component of penicillin acylase, (e) where the DNA sequence according to (d) optionally a Promoter or a promoter / leader can be connected upstream, (f) where the DNA sequence can be obtained from ATCC 11105, and DNA sequences analogous to (d) which can be obtained from microorganisms, in particular E. coli strains, penicillin acylase produce, these analogous DNA sequences optionally having the feature (e). 3. DNA sequence, characterized by the following features: (g) a DNA sequence (DNA sub-sequence) according to claim 1 (a), (h) a DNA sequence (DNA sub-sequence) according to claim 2 (d) _s which (read from AGC) follows the DNA sequence according to claim 1 (a); (i) that the DNA sequence (DNA sub-sequence) according to claim (a) optionally behind a promoter or promoter / leader can be switched according to claim 1 (b), which is responsible for both DNA subsequences, -3- as well as DNA sequences which are DNA sub-sequences analogous to (g) and (h) include and can be obtained from microorganisms, in particular E. coli strains, the penicillin acylase produce, these DNA sequences comprising analogous DNA sub-sequences optionally exhibiting the feature (i) eat. 4. Clonable DNA structures (vectors), characterized in that they have a DNA sequence according to Claim 1, 2 or 3 included. 5. Process for the production of penicillin acylase, thereby characterized in that one - of a DNA sequence according to claim 3 with the features (g) and (h) and obtainable from ATCC 11 105 the promoter or promoter / leader according to the following scheme JT C T T T T'A G A TJC A C A T T A A T Gl AAATTTTTG TIA TCAAAAAT TIA GTTATCGCO IC TCACAGTT CIA TAATGAAAC SA ATTCTCTG CIA Ä ATAGATA AIA CCG ₁ AAGCT TIC —4— IGTTGCT AGTAlTC A ATTCGC TIA_A.T_T ATACAC! -Leu- -Pro- -GIu- -Asp- -Thr- -Met- -Las- -Asn- -ArS- -Asn- ! CTGCCAGAG G! ATACAftTGAASAAATAGAAAT! End of promoter γ start of leader MEt ILe VAl ASn CYs VAL THR ALa SEr- CGTATGATC GiT GAACTGTG T! T ACTGCTTC C! -LEu MEt TYr Tar TRp Ser Leu Pro- -al * leu β1 - tC TGATGTAT TSA TTGGAGCT TIA CCTGCACT GiG C T exchanged for another promoter or promoter / leader and - Can form a prokaryotic microorganism penicillin acylase with the aid of the exchange product. 6. A process for the production of penicillin acylase, characterized in that that DNA sequences are used in the process which are obtained from microorganisms, in particular E. coli strains which produce penicillin acylase, and the DNA sequence according to claim 5 with the features (g), (h) and (i) are analogous, - With the promoters or promoters / leaders of the obtained Exchanges DNA sequences for other promoters or promoters / leaders and - With the help of the exchange products, a prokaryotic microorganism penicillin acylase can be formed. -5- 7. Process for the production of penicillin acylase, thereby marked that one - The DNA sequence according to claim 1 (a), which only contains the approx. 20 kD subunit encoded, switched behind an optionally inducible promoter or promoter / leader, - The DNA sequence according to claim 2 (d), which codes only the approximately 66 kD subunit, after one if necessary inducible promoter or promoter / leader switches and - the two subunits of a prokaryotic microorganism can form (each individual of the microorganism forming both subunits) and optionally extracted or - the two sub-units for. separate in different individuals of a prokaryotic microorganism can form, optionally extracted and, if appropriate, then combined and - If necessary, the penicillin acylase formed is then isolated. 8. A process for the preparation of penicillin acylase, characterized in that one - a DNA sequence that has been obtained from a microorganism, in particular an E. coli strain, the penicillin acylase produced, and which is analogous to the DNA sequence according to claim 1 (a), behind an optionally inducible Promoter or promoter / leader switches, - a DNA sequence that has been obtained from a microorganism, in particular from an E. coli strain, the penicillin acylase and which is analogous to the DNA sequence according to claim 2 (d), after an optional inducible promoter or promoter / leader switches and - the two subunits of a prokaryotic microorganism can form (each individual of the microorganism forming both subunits) and optionally extracted or -6- - the two subunits separately in different Lets individuals of a prokaryotic microorganism form, optionally extracted and optionally then gives together and - If necessary, the penicillin acylase formed is then isolated. 9. The method according to claim 8, characterized in that the two DNA sequences that each code for one of the two subunits of penicillin acylase, obtained from one and the same microorganism Has. 10. The method according to claim 7, 8 or 9, characterized in that the promoter or promoter tor / leader uses the DNA sequences obtained from ATCC 11105 according to claim 5. 11. The method according to any one of the preceding claims, characterized in that one is used as a prokaryotic Microorganism uses a strain exhibiting protease activity equivalent to that of E. coli 5K or larger, for example DSM 1454.